Health Problem Well over 1.1 million in the U.S., and 35.5 million people globally, are infected with HIV. With highly active, or combination, anti-retroviral therapy (HAART/cART), HIV-AIDS becomes a chronic condition. Unfortunately, HAART is not a cure; if treatment is stopped, HIV virus production rapidly rebounds and leads to full-blown HIV-AIDS. HAART fails to eradicate HIV infection because it has little effect on latent cells carrying replication- competent HIV genome copies, and it fails to fully control HIV replication in the central nervous system (CNS), oral and intestinal mucosa. Issues with Current Solutions & How Product Meets Unmet Needs A recent NIAID contract funds the expansion of laboratories offering Q-VOA to evaluate novel therapies to activate or eradicate latent HIV-infected reservoirs in HAART patients and to validate novel assays of latent and persistent HIV infection. The currently accepted method of quantifying the latent reservoir is the quantitative viral outgrowth assay (Q-VOA), which is resource and time intensive and underestimates the HIV latent reservoir. The NIAID Q-VOA Resource contract confirms a dire need to expedite the validation and availability of assays that more accurately quantitate latent HIV reservoirs in HAART patients. However, the currently available VQA and WHO HIV-1 virus standards are not relevant controls for cell-based HIV-1 RNA detection assays, creating a critical bottleneck in the Q-VOA cross-validation process. Further, Armored RNA(r) control kit used within the Abbott and Roche RT-PCR diagnostics for HIV virus particle quantification is based on the inherently flawed MS2-VLP technology. Summary of Approach The goals of the Specific Aims of this Phase I proposal are to develop and validate a novel, marine-based virus-like particle (VLP) to encapsidate cell-based HIV-1 RNA as quantitative standards for latent HIV-1 RNA detection assays. The novel VLP HIV-1 RNA controls will be calibrated and standardized through in solution biophysical analysis and within an RNA-based assay against HAART patient samples and the VQA and WHO HIV-1 viral standards. The Phase I results will lay the groundwork for Phase II full validation and production of a market-ready, calibrated cell-based HIV-1 RNA external and internal standards kit suitable for high- throughput quantification of latent HIV-infected reservoirs in HAART patients in laboratory and clinical settings. Collaborators and Unique Resources To accomplish its Phase I aims, Jan Biotech, Inc. will collaborate with Dr. Susan Kreuger at National Institute of Standards and Technology (NIST), Dr. Amneris Luque and University of Rochester Center for AIDS Research (UR CFAR), Dr. David Margolis of the Collaboratory of AIDS Researchers for Eradication (CARE), and Dr. James Bremer of the VQA Laboratory. Dr. Kreuger was a key member of the team that used in solution biophysical methods to standardize virus-like particle (VLP) for encapsulation of RNA standards. UR CFAR will provide HAART patient samples for testing the VLP HIV-1 RNA standards. Jan Biotech will work with the VQA Laboratory to perform side-by-side testing of the novel HIV-1 RNA VLP standards against the currently used VQA and WHO HIV-1 viral standards. Phase I Specific Aims Specific Aim 1: Construction of Novel HIV-1 RNA Virus-Like Particle (VLP) Expression Vectors for Use as Standards in a Latent HIV-1 Diagnostic Kit Specific Aim 2: Quantitative Validation of the Latent HIV RNA VLP Controls by Biophysical Analysis Specific Aim 3: Testing and Analysis of the Prototype Latent HIV Control Kit How Anticipated Results will Justify Phase II and Further Product Development The Phase I is expected to produce a kit of cell-based HIV-1 RNA VLP standard controls, with proven utility in a latent HIV-1 RNA assay and preliminary validation against the established VQA and WHO standards. In the Phase II, the latent HIV diagnostic control kit will be fully standardized and validated, with the end goal of standard reference material (SRM) control kit for latent HIV-1 RNA assays.